Latex of polyhydroxyalkanoate

ABSTRACT

A chiral (R) stereospecific polyhydroxyalkanoate (PHA) in aqueous latex form having virgin disperse phase particles of very low or undetectable crystallinity is useful in making a water-sensitive structure water-resistant. It may be used as a mixture with a latex of a synthetic polymer. Processes for making the PHA latex are disclosed.

This invention relates to particulate polyester, to polyhydroxyalkanoate(PHA) in preferred forms and processes for making it, and to a structurehaving PHA as coating or binder.

PHA, the general formula of which is set out below, is accumulated bymany micro-organisms, particularly bacteria, for example of the generaAlcaligenes, Athiorhodium, Azotobacter, Bacillus, Nocardia, Pseudomonas,Rhizobium, and Spirillium, as an energy reserve material. It isconveniently prepared by cultivating the micro-organism in an aqueousmedium on an energy and carbon source. At least part of the cultivationis preferably conducted under limitation of a nutrient essential forgrowth but not required for PHA accumulation. Examples of suitableprocesses are described in EP-A-15669 and 46344.

Such PHAs containing both 3-hydroxybutyrate (HB) units alone or with3-hydroxyvalerate (HV) units are available commercially. Such PHAscontaining larger repeating units have been described.

In the paper by Marchessault et al. (Report of NATO Advanced ResearchWorkshop at Sitges, Spain 26-31 May 1990, `Novel biodegradable microbialpolymers; ed. Dawes, publ. Kluwer) there is disclosed a latex of a PHA,namely polyhydroxybutyrate-co-valerate (PHBV 21% V) supplied by apredecessor of the present applicant in which x-ray diffraction showssome crystalline diffraction in the PHB lattice. Further relevant workby this author along with various co-authors is described in otherpublications, including Polymer 1992, 33(A), 823-827; FEMS MicrobiologyReviews 1992, 103, 299-310; TAPPI Journal 1993 May, 76(5), 71-77. Fromthese it appears that the PHA, whether in the commercially obtained 21%or 27% V latex or in latex made by the hypochlorite route, was 95% pureand the latex particles invariably comprised an amorphous core within acrystalline shell. Experiments using the latex in making films aredescribed and the suggestion is made to use it as a binder, coatingmaterial or barrier for paper or non-wovens. In patent application WO91/13207 (corresponding U.S. Pat. No. 5,451,456) of the same authoralong with co-inventors the use of the 21% V latex in the production ofthe film and coated paper is described in more detail and a content ofamorphous polymer is designated as essential.

Co-pending application WO 95/15260 describes a composite film made byapplying partly amorphous, for example 34% crystalline, PHBV as latex topreformed cellulose film.

Co-pending application WO 94/07940 describes the preparation of a PHAsuspension containing NPCM (non PHA cell material) decompositionproducts and surfactant, in which 60% of the PHA particles are amorphous(density 1.176) and 30% are crystalline (density 1.231), the remainderbeing non resolved in caesium chloride gradient solution. No means ofremoving the dissolved materials from the suspension is disclosed.

In each of the above prior documents the PHA may be `virgin`, that is,the product of solubilising and removing NPCM leaving the PHA particlesas laid down in microorganism cells or as agglomerates thereof. (Whereasit is possible to make a latex by dissolving PHA in a volatile liquidsuch as chloroform, emulsifying the solution in water and removing thevolatile liquid e.g. by evaporation, the PHA particles of such a latexare not virgin).

We have now prepared latex in which the suspended particles are ofhigher amorphous content. Such a latex has interesting properties forexample substantial advantages when used in conjunction with film, paperor non-wovens, the advantages lying in the quality of the productsand/or the convenience of the procedure used in making them.

The invention in its first aspect provides an aqueous latex ofmicroobiologically produced PHA comprising virgin disperse phaseparticles of which fewer than 30, especially fewer than 20, especiallyfewer than 1, % w/w are crystalline.

Percentage crystallinity is as measured by density, X-ray diffraction orsmall or wide angle x-ray scattering (SAXS or WAXS). The percentages areby weight and are believed to represent: ##EQU1## where each particle iseither wholly amorphous or at its maximum attainable crystallinity.

The particles of PHA are of weight average diameter (d 50) preferablyunder 5 μm, for example in the range 0.05 to 1.5 μm. Latex havingparticles of weight average diameter in the range 0.4 to 1.1 especially0.5 to 1.1, μm is preferably the product of the process describedhereinafter as the fourth aspect of the invention. Latex havingparticles of weight average diameter in the range 0.05 to 0.5 especially0.1 to 0.4 μm is suitably the product of the process describedhereinafter as the third and fourth aspect of the invention.

Suitable PHAs comprise repeating units of formula I:

    --O--C.sub.m H.sub.n --CO--                                I

where m is in the range 1-13 and n is 2 m or (except when m is unity) 2m-2. Typically C_(m) H_(n) contains 2-5 carbon atoms in the polymerchain and the remainder (if any) in a side chain. In very suitable PHAsm is 3 or 4, n is 2 m and especially there are units with m=3 and m=4copolymerised together with respectively a C₁ and C₂ side chain on thecarbon next to oxygen. The molecular weight of the PHA is for exampleover 50000, especially over 100000, up to eg 2×10⁶.

PHA of formula (I) containing only m=3 units is referred to as PHB; PHAcontaining m=3 and m=4 units is the co-polymer PHBV. The PHBV preferablycontains 4-50, especially 4-30 and more especially 10-30 for example12-25, mol % of m=4 units. The PHA can also be a blend of two or morediffering in the value of m. A particular example contains:

(a) PHA consisting essentially of Formula I units in which 2-5 mol % ofunits have m=4, the rest m=3; and

(b) PHA consisting essentially of Formula I units in which 15-30 mol %of units have m=4, the rest m=3. The proportions in such a blend arepreferably such as give an average m=4 content in the range 12-25%. Toarrive at such blends, latices differing in m=3 and m=4 content, orprecursors thereof, are mixed. However, it is preferred to use singlepolymers or blends of polymers all within the m=4 range 10-30 mol %.

The PHA is the product of a microbiological process in which themicroorganism may be wild or mutated or may have had the necessarygenetic material introduced into it. Alternatively the necessary geneticmaterial may be harboured by a eucaryote, to effect the microbiologicalprocess. Microbiologically produced PHA is chiral (R) andstereospecific.

Examples of suitable microbiological processes are the following:

for Formula I material with m=3 or m=partly 3, partly 4: EP-A-69497(Alcaligenes eutrophus);

for Formula I materials with m=3;

U.S. Pat. No. 4,101,533 (A. eutrophus), EP-A-144017 (A. latus);

for Formula I material with m=7-13: EP-A-0392687 (various Pseudomonas).

Usually such processes include a cell growth stage followed by aPHA-accumulation stage: in the latter, an assimilable carbon source isprovided, but the concentration of one or more nutrients essential tocell growth is limited or maintained at substantially zero. Suchnutrients are conveniently one or more of nitrogen, phosphorus, sulphuror magnesium. The latex according to the invention is preferably theproduct of accumulation under limitation or absence of phosphorus.

The PHA is then separated from the PHA-containing cells by a harvestingprocess as described further below.

The polymer content of the latex is, in total if more than one ispresent, in the range 10-60, especially 20-50,% w/w.

The latex preferably contains a stabilising quantity of a surfactantother than NPCM.

The stabilising quantity of surfactant is typically in the range 0.25 to10, especially 1 to 7% w/w on PHA dry solids. This is or includessurfactant in the latex after all steps of NPCM removal have beencomplete, including the final step of removing soluble NPCMdecomposition products and unadsorbed surfactant. Thus it may correspondto a monolayer on the surface of the PHA particles, so far as this canbe inferred from the sizes and shapes of the particles. Typically thisquantity is the residue after washing of the surfactant used in aprocess according to the fourth aspect of the invention.

The surfactant can be anionic, cationic, non-ionic, zwitterionic orcontain hydrophilic groups of more than one type. The hydrophilic partof the surfactant preferably contains at least 8, especially 12-20,carbon atoms per hydrophilic group. It may be (almost) wholly aromaticas in sulphonated naphthalenes and naphthyl methanes; or partly aromaticas in alkyl benzene sulphonates or ethoxylates; or wholly aliphatic.Very suitably the surfactant contains a linear alkyl group. If thesurfactant is cationic, preferably its hydrophilic part is quaternaryammonium, based for example on tri C₁ -C₄ alkylammonium. If it isanionic, the hydrophilic group is typically sulphate, sulphonate,carboxylate, phosphate or phosphonate. If it is non-ionic, it may be forexample an ethoxylate, for example, an alkyl ethoxylate containing 7 to16 (especially 12 to 16) alkyl carbons and up to 100 (especially about20) ethoxylate units, or a block copolymer of ethylene oxide andpropylene oxide or an alkylphenyl-ethoxylate (especially nonyl phenolwith about 30 ethoxylate units). Suitable cationic surfactants include,typically as chloride or bromide: dodecyl-, tetradecyl- andcetyltrimethyl-ammonium, cetyldimethyl-ethylammonium, dodecyl-,tetradecyl- and hexadecyl-benzyldimethylammonium, benzalkonium,benzethonium, methylbenzethonium and cetylpyridinium. Suitable anionicsinclude, typically as sodium or ammonium salts: dodecyl sulphate,N-lauroyl-sarcosinate, dioctyl-sulfosuccinate, cholate, deoxycholate,laurate, myristate, palmitate, and stearate. Suitable non-ionics includesorbitan monopalmitate, alkylglucosides and nonyl phenyl-ethoxylates.The cationic preferred is cetyltrimethyl-ammonium bromide. Sodium orammonium deoxycholate, dodecyl sulphate, N-lauroylsarcosinate anddioctylsulfo-succinate are preferred as anionics. Ammonium forms of theanionics are preferred when it is desired to avoid introducing mineralmatter.

Instead of or in addition to using such surfactant, the latex maycontain an agent providing steric stabilisation, for example at leastone water-soluble copolymeric dispersant containing a plurality--atleast 2 and typically at least 10 and up to e.g. several hundred--ofrepeating units, including units of two types:

A PHA-compatible; and

B hydrophilic.

Such stabilisation is the subject of our co-pending GB application9525390.2 filed Dec. 12, 1995.

Type A units may be for example aliphatic hydrocarbon (for example as inaddition polymers) or aromatic hydrocarbon or (in chain lengthssufficient to give water-insolubility in a corresponding polymerconsisting of such units) polyoxyalkylene, especially poly-1,2-propyleneoxide or polyester of the head-to-tail or heat-to-tail/tail-to-headtypes such as for example, 12-hydroxy stearic acid polycondensate oralkyd resin. Preferably type A units carry substituents such asesterified carboxy groups or esterified or etherified hydroxy groups orboth, since these afford greater compatibility with the PHA and arecharacteristic of compounds effective as plasticisers for PHA.Particular examples of such substituents are disclosed below for latexaddition polymers as in the above-mentioned surfactants, and there maybe hydrophilic groups of more than one chemical composition or ioniccategory. Very suitably it is polyethyleneoxy, especially 10 to 100ethylene oxide units long, as is typical of conventional water-solublesurfactants.

The dispersant may contain a minor proportion for example under 20 molpercent, of units falling into neither type A nor type B.

The balance of type A and type B units should be such as to provide thewater solubility, which typically is at least 1% w/w in water at 20° C.Preferably the type B units are in a minority by moles, for example lessthan one-third of the total chain units in the copolymer chain;correspondingly the water-soluble portion of the type B units, ifpolyethyleneoxy, should be sufficiently long. The HLB number (HLBsignifies hydrophile-lipophile balance rating) of the dispersant issuitably in the range 10-15. Generally the dispersant is preferably fromthe class of non-ionic emulsifiers.

The latex may be formulated with additives such as:

(a) further, possibly different, surfactant;

(b) water soluble polymer as thickener and/or stabiliser, for example,cellulose ethers, vinyl or acrylic polymers, xanthan gum and associativethickeners eg based on urethane, acrylic or cellulose polymers;

(c) biocides, for example, 1,2-benzisothiazoline-3-one;

(d) co-solvents, for example, partly esterified or partly etherifiedglycols;

(e) pigment and/or pigment dispersant;

(f) one or more plasticisers;

(g) inorganic filler, for example glass fibre, carbon fibre, platy orfoil particle, silica, clay, magnesium silicate;

(h) organic filler, for example cellulose fibre or particulate, proteinfibre, synthetic polymer particle or fibre, wood flour;

(i) latex of polymer other than PHA for example as described below.

The latex is preferably substantially free of trace nutrient cations,for example those of manganese, iron, copper and zinc. It is alsopreferably very low in other soluble salts, other than the counter-ionsof surfactants. Very suitably it is the product of a process in presenceof a chelator.

The invention provides also a mixture of the above-defined latex withother latices, for example a different PHA or of a wax or naturaladdition polymers such as rubber or synthetic polymers such as PVC,PVDC, SBR, polyvinyl-acetate and polyacrylic esters as hereinafterdefined. Further details are given hereinafter, in the sixth aspect ofthe invention. A particular mixed latex is binodal in particle sizedistribution especially with one maximum at about 0.21 μm and another atabout 1 μm. The relative proportions may be such that the smallparticles substantially fill voids between the large ones, when thelatex is subjected to coalescence.

The invention provides a harvesting process for making the latex from asuspension of microbiological cells containing the PHA by the steps ofoxidatively solubilising at least partly the NPCM and applying to theresulting PHA particles a stabilising quantity of surfactant before thePHA has crystallised to the extent specified.

In this process the NPCM is solubilised leaving PHA of high purity,preferably at least 96, especially at least 98, % w/w. The harvestingprocess preferably includes a step of NPCM decomposition oxidation byperoxide in presence of a chelator and a surfactant, as describedhereinafter as the third and fourth aspects of the invention.

Whichever sequence of steps is used in making the PHA virgin latex, anysuspension that has undergone any NPCM attack should not be stored formore than a few days before receiving surfactant or undergoingoxidation. For example 7 days' storage of such a suspension has beenobserved to effect a halving of amorphous content.

Further precautions desirable for avoidance of crystallisation includekeeping the suspension at a low ionic strength, keeping the suspensionaseptic, not using hypochlorite and keeping the suspension within 2 pHunits of neutrality. To keep down the ionic strength the production ofthe biomass is preferably in chemostat conditions maintaining the pH byaddition of alkali in response to electrochemical measurement.

The invention in its second aspect provides a process of coating asubstrate, more particularly making a water resistant structure, byapplying to a water-sensitive substrate an aqueous PHA latex accordingto the first aspect. (The first aspect has been defined to includepreferred latices and mixtures described hereinafter and the product ofprocesses so described).

The substrate comprises at least sufficient water-sensitive material tobe disintegrable in presence of water, whether liquid or vapour, withoutor with the action of acid, alkali or microbiology. In the first,simplest, case it consists of material soluble in liquid water: examplesare non-polymeric substances, water-soluble cellulose ethers, polyvinylalcohols, (possibly partly acetylated) and starch including foamedstarch. In a second case it consists of materials swollen by water butnot dissolved; examples are regenerated cellulose and incompletelyacetylated celluloses. In a third case it consists of elements, such asfibres or plates or granules, bound together by such soluble orswellable materials or by inorganics such as alkali silicate. In afourth case the substrate may consist of or be bound together by abiodegradable polymer such as a microbiologically produced polymer (egPHA for rapid degradation) or a synthetic (e.g. polylactide orpolycaprolactone for moderately rapid degradation).

Particular examples of the first and second cases are cereals and cerealproducts (especially breakfast cereals and wafers), sugar, sugarconfections (especially chocolate), fruit (for example dried fruitespecially raisins), meat and fish (raw, preserved or cooked), and alsochemicals or medicines. Water insoluble packages for materials in thesecases may be improved e.g. in moisture exclusion, by a coating of PHAsand/or mixtures described herein.

Particular examples of the third case are paper, fibre board andnon-wovens. The invention is especially applicable to these, since itcan afford a moisture barrier without rendering the coated materialnon-disposable by composting. Articles of this case include packagingsheets and containers, personal hygiene products, waste disposal bagsand films for agriculture or horticulture. In view of the high glossattainable, articles of good customer appeal can be produced, especiallyfor food service crockery, including fast food packages and insulatingpackages. When used as a binder for fibres such as the cellulose fibresof paper or non-wovens, the PHA affords a substantial increase inmechanical strength. This property has not been observed using laticesof PHA of a higher level of crystallinity.

Materials of the third and fourth case can be subjected to a degree ofbiodegradation and/or hydrolysis sufficient to permit re-use of atleasst part thereof. For example a PHA coating, possibly as a componentof printing ink on fibre board can be biodegraded or solubilised and theboard binders softened or dissolved sufficiently to permit pulping suchas in a beater machine.

The latex may be applied to the substrate for example by dipping,spraying, doctoring, gravure-roll coating or reverse-roll coating. Theapplication may be continuous over the surface, or through theinterstices, of the substrate: if desired, it may be patterned, for adecorative effect or to effect a differential rate of biodegradation forexample as between areas having PHA and uncoated areas, or with thicklycoated areas alternating with thinly coated areas. Whichever applicationmethod is used, it is preferably controlled so that the appliedparticles are at least partly amorphous, although they may be less sothan in the latex.

After application, the particles may be treated to effect coalescence.This can be effected by exposure to solvent vapour. Preferably it iseffected by heating, for example in hot gas or in contact with a hotsurface such as a calender-roll or by radiation such as microwave orinfra-red. The temperature need not be high enough to melt the PHA.Preferably the heating to effect coalescence is applied to the latexcarrying substrate while still wet, that is, while the latex componentstill contains water to the extent of at least 20 especially at least50% w/w on the PHA. Thus drying and coalescence take place in a singlestep. For such a single step infra red heating is especially suitable.The single step of drying+coalescence can be effected in 10 to 20seconds. This makes possible a continuous process of passing acontinuous web substrate or continuous succession of substrate piecesthrough an application zone followed by an infra-red heating zone, withlittle or no drying between the zones and with infra-red input adjustedto complete drying and coalescence in a residence time under 30 seconds.

Whereas in general coalescence of the latex particles is affected byheating in preference to the action of solvent vapour, the heat inputrequired can be less and the operating temperature lower than when usinglatex having a substantial content of crystalline or part crystallinePHA. When the particles are of average diameter less than 0.7 μm, e.g.in the lower ranges herein, heat input need be little more than, e.g. upto 150% of the latent heat of evaporation of the water of the appliedlatex coating. As a result, good coalescence is obtained in latexcoating with drying at ambient temperature (15-25° C.). For more rapidprocessing, the temperature can be up to 50° C. or even 100° C. or thecoated substrate can be dried at reduced pressure. The effect of usingsuch small-particle latex is thus to permit the use of low-grade heatsources and/or the coating of temperature sensitive substrates. Suchlow-temperature processing is available whether the latex is virgin, asdescribed elsewhere in this specification, or is in the product ofdissolving the PHA in a water-immiscible liquid such as chloroform or1,2-dichloroethane, emulsifying the resulting solution in water andremoving the liquid, or of dispersing PHA in water as a melt or asolution in a water-soluble liquid.

Accordingly, as a modification of the second aspect a process of coatinga substrate, e.g. for making a water resistant structure by applying aPHA latex thereto is characterised by using a latex in which the PHAparticles are of average particle diameter up to 0.7 μm and drying theresulting coated substrate with a heat input little more than the latentheat of evaporation of the water applied in the latex.

Apart from the particle diameter and heat input features, the preferredconditions for this process are as described elsewhere herein.

Preferred products of the process or the modification thereof includepaper and board carrying a glossy PHA coating, whether or notcalendered, especially those characterised by the features:

(a) cellulose fibre paper or board substrate 20 to 600 g per m² ;

(b) PHBV (0 to 30 mol % V) coating, 0.5 to 30 μm dry thickness;

(c) gloss value 65-100, especially 70-75% at 60 degree incident lightangle.

These values are substantially equal to those obtained by melt-coatingthe PHA. Such paper or board may carry the PHA layer on one or bothsides.

The invention provides a fabric construct such as a paper or non-wovenfabric having a PHA latex in its interstices and a construct of highmechanical strength made by heating such a construct so as tocrystalline the PHA. The PHA latex is preferably as herein described.

The latex is also useful as a constituent of aqueous compositions to beapplied to non-absorbent surfaces.

The invention in its third aspect relates to a process for recoveringPHA in latex form from the cells in which it is accumulated.

Separation of PHA from NPCM has presented a long-standing problem.Extraction by solvent is inconvenient because of the high viscosity ofthe PHA solution and the expense of solvent recovery; it also requirescomplicated further processing steps if fine particles of PHA arerequired. Chemical treatment of the NPCM has been operated industriallybut is susceptible of improvement to control the particle size of thePHA and/or its purity.

In an early proposal (Williamson et al. J Gen. Microbiol. 1958, 19,198-203) the NPCM was solubilised by reaction with hypochlorite;however, this was accompanied by a serious loss in PHA molecular weight,for example from 10⁶ to 1.01×10⁵. (See EP-A-145233, Example 18). Refinedforms of hypochlorite treatment have been proposed (Ramsay et al.Biotechnology Techniques 1989, 3 (4), 227-232; 1990, 4, 221-226 and U.S.Pat. No. 5,110,980), but process conditions are narrowly described andappear to require extremely careful control to obtain high PHA puritywithout serious loss of yield or decrease of molecular weight. Neitherof these references discloses the production of a latex or of materialhaving a low degree of crystallinity.

Other oxidative treatments of NPCM have been proposed. According toEP-A-145233 the PHA containing cells are subjected to heat-shock andenzyme action and the resulting product is treated with hydrogenperoxide. According to application WO 94/24302 the efficiency ofoxidative treatments of NPCM is decreased by side reactions catalysed bytraces of the transition metal compounds added as nutrients in growingthe microorganism cells in which the PHA is laid down. Therefore it isproposed to carry out the oxidative treatment in presence of a chelator.According to Examples 1-4 of that application, a PHBV purity of 99.5% isobtained by treating PHA-containing cells with hydrogen peroxide and achelator. According to Example 4 the cells need not be the preliminarilyheat shocked or enzyme-treated. The product of the oxidation treatmentis processed to dry PHA.

A further expedient proposed for NPCM removal is treatment with asurfactant. For example in Example 5 of EP-A-145233 it is reported thatPHB made by boiling cell suspension at 100° C. for 60 min was 72% purein absence of added surfactant but 93% pure in presence of sodiumdodecyl sulphate (SDS) 10% on cell dry weight. In Ramsey et al(Biotechnology Techniques 1990, 4, 221-226 and U.S. Pat No. 5,110,980)it is disclosed that biomass that has been treated with anionicsurfactant and separated therefrom is more fully freed of NPCM byhypochlorite digestion. In co-pending application WO 94/10289 it isdisclosed that PHA bearing cells were treated with SDS and hydrogenperoxide for 16 hours at room temperature, washed and treated withhydrogen peroxide at 80° C. for 3 hours; the resulting PHA was at least99% pure. However, the product PHA was processed to dry polymer.

Much work has been done on enzymatic degradation of NPCM. Thus inEP-A-145233 a proteolytic enzyme, such as "Alcalase", "Protease","Neutrase", "Esparase", "Allprotease" or "High T" (all believed to beregistered trade marks) or bromelain or papain and/or combinations oftwo or more of these, were applied to cells following a "heat-shock"treatment at 80-200° C.; the product of such proteolysis may besubjected to a phospholipase enzyme for example "Lecitase" or to otherenzymes exemplified by lysozyme and "Novozyme" (RTM). WO 94/07940describes the preparation by the succession of heat shock, thenprotease, then surfactant+hydrogen peroxide of a PHA suspension in which60% of disperse phase particles are amorphous, 30% are crystalline and10% were not resolved. These particles have a mean diameter (d 50) of0.98 μm.

A latex of virgin PHA particles of d 50 weight average diameter mainlyor wholly in the range 0.05 to 0.5 μm, and mainly or wholly amorphoushas now been produced. It has valuable properties such as rapidcoalescence when used to produce films or coatings.

According to the invention in its third aspect an aqueous latexcomprises PHA in the form of substantially amorphous wet virginparticles that are single or agglomerates of up to 25 single particles.The particles are of average diameter d 50 in the range 0.05 to 0.5,especially 0.1 to 0.4, μm and preferably are substantially free ofcontamination by alkali metal ions or halogen ions or organic halogen orchelatable metal ions. They are preferably less than 30, preferably lessthan 20 and especially less than 1, percent crystalline, measured ashereinbefore described.

By "single" is meant the particles as they existed within the cell wallsof the microorganisms in which they were laid down. It appears thatwithin the walls of each cell of the microorganisms listed above,especially those mentioned below, the PHA is laid down discretely as anumber--typically up to a few hundred, for example 10 to 25--of suchparticles. It is believed that the particles according to the inventionare these single particles or intra-cell agglomerates thereof. Typicallythey contain less than 4, especially less than 2, % w/w of NPCMresidues.

The latex may be the product of separating PHA from cells containing itby a harvesting process including NPCM solubilisation by peroxide in thepresence of a chelator and a surfactant, especially as described below.

The surfactant may be present as a result of use in separating NPCM, butthere may be present instead or in addition, stabilisers such as othersurfactants and/or water-soluble polymers. Examples of these and otheradditives are set out above.

The concentration of PHA in the latex is typically 100 to 600,especially 200 to 500, g/l.

The concentration of surfactant in the latex is as described for thefirst aspect of the invention.

The invention also includes a latex precursor, that is, beforeseparation of some or all of the NPCM decomposition products andunadsorbed surfactant if present. Typically the surfactant concentrationin such a latex precursor is in the upper two-thirds of the rangesmentioned above.

In its fourth aspect the invention provides a process for making PHA byproducing by fermentation a biomass of microorganism cells containingPHA and harvesting PHA by decomposing NPCM by steps including treatmentwith a surfactant and an oxidant: characterised in that the saidtreatment is applied to the biomass before substantial (as hereinafterdefined) decomposition of the NPCM by other means.

A preferred process is characterised further in that the biomass towhich the treatment is applied is the product of fermentation underphosphorus limitation.

The treatment is applied to the biomass preferably without significantdilution or concentration (that is, by a factor of more than 2),conveniently to whole fermentation product, which typically contains100-200 g/l of cells (dry weight excluding PHA) the cells containing50-80, especially 65-75, % w/w of PHA.

The oxidant is preferably one that at pH levels up to 1 unit above orbelow neutrality is stable against side reactions. It is preferably aperoxide, especially hydrogen peroxide and preferably a chelating agentis present, for example an amino polycarboxylic acid such asethylenediamine tetra acetic acid or a nitriloacetic acid, a hydroxypolycarboxylic acid such as citric acid or tartaric acid; an aminopolyphosphonic acid such as diethylene triamine penta methylenephosphonic acid (DTPA); or a polyphosphate such as tripolyphosphate.Each is of course present as a salt corresponding to the pH to which themixture is adjusted. The temperature of the oxidation step is suitablyin the range 50-90° C. and this is typically maintained for 6-24 h.

The surfactant can be any of those listed above; if two or more are usedtogether, these should normally not include both a cationic and ananionic. The content of surfactant during this step is suitably in therange 0.2 to 10, especially 1 to 5, % w/w on dry PHA solids.

Preferably the oxidant, chelator and surfactant are present together forat least the bulk of the reaction time. Preferably the chelator andoxidant added are before the surfactant, or before the temperature israised to 50° C. or above: thus surfactant may be present as the resultof a pre-treatment at under 50° C., as described further below. To limitside reactions, the chelator is preferably added before the oxidant. Thetemperature and time can be in the same range as defined above for theoxidation step.

The harvesting process normally includes separation of PHA from solubleNPCM decomposition products; this step also separates surfactant. WhenPHA is to be recovered as a latex the quantity thereof used forsolubilising NPCM is preferably greater than the quantity needed tostabilise the latex. In the separation step it is important to avoidmechanical treatments sedimenting or compressing the particles togetheror otherwise injuring them. This is the subject of the fifth aspect ofthe invention.

By "substantial decomposition" of the NPCM is meant over 50% w/wsolubilisation of more than one of three major components of NPCM,namely protein, nucleic acid and peptido-glycan. If a treatment stepbefore the claimed surfactant and oxidant treatment is used, it ispreferably at not over 50° C., for example:

(a) mechanical homogenisation suitably at high pressure using forexample a French pressure cell and possibly in presence of surfactant;

(b) oxidant/surfactant depolymerisation of nucleic acid; or

(c) enzyme digestion to solubilise peptido-glycan by a cell wall lyticenzyme such as lysozyme. In general the process may include other stepspreviously proposed for such harvesting, for example heat-shocking thecells, cell wall breaking by audible sound, ultra-sound or high pressurehomogenisation or even freeze-drying providing that substantialcrystallisation or agglomeration is not provoked thereby.

The step of cell wall disruption by mechanical means in presence ofsurfactant is of more general application and constitutes a furtheraspect of the invention.

Whereas the invention in the fourth aspect is primarily concerned with alatex and latex-making process, it may include the further step ofconverting the treated biomass or the latex to larger PHA particles or(via such particles or direct) to dry PHA. The surfactant should be ofsuch a kind or in such a concentration that the properties of articlesto be made are not impaired.

Conversion to larger PHA particles is conveniently effected by heating,as described in co-pending Application WO 94/02622. Conversion to dryPHA can be effected by spray-drying. Other preferred methods, includingfreezing, freeze-drying or flaking, also produce particles of highaccessible surface, so that excess surfactant or stabilising colloid canbe removed by washing. Such conversion can be carried out in conditionsgentle enough to avoid gross crystallisation of the PHA; this of courseis not critical if subsequent solution- or melt-processing is intended.

The PHA can be used in any of the usual methods for forming shapedarticles from dry polymers, for example, injection moulding, extrusionincluding co-extrusion with other polymer, spinning or coating ontosubstrate.

The fifth aspect of the invention arises from the observation that manyprocedures for removing such dissolved materials, for example bowlcentrifugation or filtration, provoke a substantial increase incrystallinity.

According to the fifth aspect of the invention a process for producing aPHA latex of substantial amorphous content comprises:

(a) forming a biomass containing micro-organism cells containing PHAparticles;

(b) decomposing the non-PHA cell material of the cells to solubleproducts;

(c) separating the soluble products from the PHA particles; and

(d) forming an aqueous dispersion of PHA particles substantially free ofsuch soluble products; and is characterised by:

carrying out step (c) by resolving the product of step (b) into asolution of the soluble products and a non shear-thickening slurry.

The slurry is flowable and contains preferably not more that 50, forexample in the range 10-40,% w/w of PHA particles.

Such resolution distinguishes particularly from conventional filtrationin which filtrate is squeezed from a wet filter cake, or conventionalcentrifugation in which a static pellet or layer is formed and subjectedto enhanced gravity to remove liquid; such conventional procedurestypically also include mechanical working to effect redispersion in aidof further washing or to produce a latex. The process of the inventionis preferably carried out without, or with at most transient orlocalised, formation of paste more concentrated than the stated limits.

Step (b) may include initial stages possibly in absence of addedsurfactant. These stages typically include one or more of:

heat-shock, that is, a rapid temperature rise to 80-200° C. effected forexample by injection of high pressure steam;

enzyme treatment by hydrolase such as lysozyme to decompose peptidoglycan;

enzyme treatment by a proteolytic enzyme to decompose cell wall protein,

cell wall breaking by audible or ultra-sound;

high pressure homogenisation;

oxidation.

In one preferred form of the process, step (b) is carried out by theprocess of the fourth aspect of the invention.

A process including one or more of such stages to decompose 80 to 90% ofNPCM, followed by oxidation in presence of surfactant to remove most orall of the remaining NPCM, characteristically produces particles of meandiameter in the range 0.4 to 1.5, especially 0.75-1.05 μm; by the use ofthe invention they can be under 30, especially under 20, for exampleunder 1, % w/w crystalline.

In either form of the process the solubilisation of NPCM is conducted soas to produce PHA preferably at least 96, especially at least 98, %pure.

The surfactant can be any of those listed above; if two or more are usedtogether, these should normally not include both a cationic and ananionic. The content of surfactant during step (b) is suitably in therange 0.2 to 10, especially 1 to 7, % w/w on dry PHA solids.

If desired, there may be a treatment such as mechanical treatment inpresence of surfactant or a lysozyme digestion or a surfactant/oxidationstep to decompose nucleic acids, thus decreasing the viscosity, in eachcase at a temperature in the range 15-50° C.

Separation step (c) can be carried out by any method, provided theproduct slurry concentration remains within the stated ranges.Decantation can be used without special precautions. Thus, ifcentrifugation is used, the g-value and residence time are adjusted sothat the residue is a paste, rather than a pellet; conveniently acontinuous centrifuge is used. Washing by decantation can be used, aloneor as a preliminary to a different method.

A preferred method of separation involves tangential flow filtration.This is described for example in the article by Redkar and Davis(Biotechnol.Prog. 1933, 9, 625-634). It involves contacting thesuspension under pressure with a membrane of predetermined pore size(suitably 10⁵ molecular weight cut off to 0.1 μm for the process of theinvention); preferably the suspension is caused to flow along thesurface of the membrane to exert a shear. Since a static filter cakedoes not build up, there is little if any compression of the particlesand thus agglomeration and crystallisation are minimised.

A preferred method of separation comprises:

(a) flowing the latex transversely of one side (the upstream side) of amembrane permeable to solutes but impermeable to latex particles, at aflow rate setting up a shear maintaining the PHA particles of the latexin suspension;

(b) taking an aqueous solution of the solutes from the other side (thedownstream side) of the membrane;

(c) adding further water to the suspension whereby to effect furtherremoval of solutes from the suspension.

The relative rates of solution off-take in (b) and water addition in (c)may be controlled to effect a net dilution or concentration of thestarting latex. The added water may contain other materials, for examplesurfactant, if it is desired to maintain the presence of such materialin the resolved latex. Preferably the liquid at the upstream side of themembrane is maintained at a hydrostatic pressure higher than at thedownstream side. In this method, since a static filter cake does notbuild up, there is little if any compression causing mutual collision ofthe particles and thus agglomeration and crystallisation provokingevents are minimised.

If the process includes other steps previously proposed for suchharvesting, for example heat-shocking the cells, cell wall breaking byaudible sound, ultra-sound or high pressure homogenisation or enzymedigestion, or even freeze-drying, these should be in conditions suchthat substantial crystallisation or agglomeration is not provoked.

It has been found that substantial advantages accrue when latices of adefined class are used in conjunction with the latex describedhereinbefore.

According to the invention in its sixth aspect there is provided amixture of at least one PHA latex (first latex), at least part of whichis according to the first aspect of the invention, with at least onelatex (second latex) of a polymer other than the PHA and preferablycompatible with the PHA, at least when at the low crystallinity levelsherein-before described.

The said polymer may be a condensation polymer or an addition polymer.Such an addition polymer may have at least one of the followingcharacteristics:

average particle diameter in the range 10 to 1000, especially 20 to 500,more especially 50 to 100 nm;

surfactant concentration less than 3, especially less than 2, forexample in the range 0.2 to 1.5%, w/w on total solids;

at least one-third, especially at least one-half, by moles of itsrepeating ethylene-residue units carrying direct or through CO at leastone oxygen-linked hydrocarbon group of 2 or more carbon atoms.

The particle diameter of the second latex is preferably less than, forexample 0.001 to 0.1 or up to 0.5 times that of the first latex.

The surfactant concentration of the second latex is preferably less thanthat of the first latex. It is believed that, on mixing the two latices,the second latex may take up from the liquid phase of the first latex,surfactant, leaving the first latex less tending the shear-thickening orflocculation.

The oxygen-linked residues carry ester or ether groups. Examples ofester groups are (a) those of the acids acrylic (as hereinafterdefined), maleic, fumaric and itaconic, with C₂₋₁₈ alcohols and phenols;(b) those of allyl alcohol or the notional vinyl alcohol with C₂₋₁₈carboxylic acids. Examples of ether groups are those of allyl alcohol orthe notional vinyl alcohol with C₂₋₁₈ alcohols and phenols. Suchalcohols and carboxylic acids can be straight-chain, branched or cyclicbut, if substituted, do not include groups conferring water-solubilityon the polymer in the proportion used.

The balance of the units in the second latex polymer can be for exampleone or more of ethylene, propylene, styrene (preferred), vinyl halides,vinylidene halides, vinyl methyl ether, acrylic (as hereinafter defined)nitrile or methyl ester and conjugated olefins. Hydrophilic groups suchas carboxylic acid or amide should be at too low a concentration toconfer water-solubility, typically under 10 mol %.

The term `acrylic` is hereby defined by the general formula: ##STR1##where R₁ is hydrogen, C₁₋₁₂ alkyl (especially methyl), cycloalkyl, aryl,halogen or cyano and R₂ is a C₂₋₁₈ hydrocarbon group. The analogousdefinition applies to the corresponding nitrile if present.

The condensation polymer is suitably a polyester or polyurethane.

According to the invention in its seventh aspect a process of making awater resistant structure comprises applying to a water sensitivesubstrate mixture of the said first and second latices.

The first latex may contain crystalline PHA particles but preferably theextent of crystallinity is low, as hereinbefore described. It preferablycontains only virgin particles, that is, the product of forming PHAmicrobiologically as a cell constituent, then solubilising and removingnon-PHA cell material (NPCM) leaving the PHA particles as laid down inmicroorganism cells or as agglomerates thereof. Alternatively the firstlatex may be wholly or partly the product of emulsification, that is,dissolving PHA in a volatile liquid such as chloroform, methylene,chloride or 1,2-dichloroethane, emulsifying the solution in water andremoving the volatile liquid e.g. by evaporation or diffusion, asdescribed in co-pending application WO 94/07940. When the first latex isthe product of emulsification, the PHA can be wholly or partlysynthetic, as made for example by the processes of Bloembergen et al.,Macromolecules 1989, 22, 1656-1669.

Other preferred forms of the first latex are as described in co-pendingapplication WO 94/07940, subject to the requirement that any surfactantto be present should be chosen for compatibility with the additionpolymer latex.

The first latex may be made from a solution of PHA in a liquid havingsubstantial solubility in water, for example an alkylene carbonate.

The polymer of the second latex preferably has a film forming capabilityat temperatures up to 20° C. for example in the range minus 20 to +60°C., especially 0-20° C. This polymer is preferably at least aterpolymer. Preferably no one unit of the four mentioned is present atover 50% w/w. The over-all balance of units is preferably such as toprovide amorphous resins in the "hydrophobic" class; thus at least oneof its monomers is itself substantially insoluble in water. The secondlatex is typically the product of emulsion polymerisation or dispersionpolymerisation. An organic liquid may be present.

The second latex polymer is substantially amorphous. Its molecularweight is preferably over 50000, especially over 100000. Usually itsmolecular weight is not more than 5×10⁶.

The repeating units are preferably selected from:

styrene;

acrylic esters having C₃ -C₁₀ side chains; and

acrylic acid.

The acrylic acid is present preferably to the extent of 3-10% w/w. The(meth)acrylic ester is preferably a mixture of acrylic and methacrylicesters. The acrylic ester is preferably an octyl ester; the methacrylicester is preferably a butyl ester.

The proportions of the repeating units are preferably in the ranges:

styrene x parts by weight

acrylic ester (0.7 to 1.3) x

methacrylic ester (1.5 to 2.0) x.

These relative proportions of the first and second latices can be chosenaccording to the intended use of the mixture and the technical effectdesired. If the intended use is biodegradable-coated substrates, theproportion of second latex should be limited, for example up to 20% w/won a dry solids basis, but can be higher for example in the range 20-80%w/w if biodisintegration is acceptable. If rapid biodegradability is notessential, higher contents of second latex for example up to 95% w/w canbe used. The mixed latex is less liable to shear-thickening and/orflocculation than the PHA latex alone.

The second latex is typically the product of polymerising olefinicallyunsaturated monomers in presence of a free-radical-generating initiatorwith appropriate heating or irradiation in an aqueous medium withconventional dispersants. Such a latex could be used as such or isolatedand redispersed. Typical dispersants are anionics such as Na, K or NH4salts of C₁₂₋₂₀ carboxylic acids, dialkylsulphosuccinates, sulphatedoils, alkane sulphonic acids, and alkyl sulphates and non-ionics such asexthoxylated fatty acids and/or amides. The dispersant can be of a typecopolymerisable with the monomers. The amount used is usually 0.1 to 5%w/w on the total monomer(s) used. Typical initiators are hydrogenperoxide, persulphates and redox systems, generally at 0.05 to 3% w/w onthe total monomers used.

EXAMPLE 1 COATING

Latices having the following properties were used:

solids content 35-40% w/w

disperse phase composition 84 HB/16 HV mol percent

molecular weight about 500000

particle size average 0.8-1 μm

percentage crystallinity under 1% by WAXS surfactant "Synperonic A20"(RTM), a condensate of C₁₂ alkanol with 20 mols of ethylene oxide.

These had been made by fermenting glucose and propionic acid withAlcaligenes eutrophus with phosphate limitation, then harvesting bysteam injection, proteolytic enzyme action and digestion withchelator+hydrogen peroxide+surfactant added in that order; solubles andexcess surfactant were then removed by membrane-flow filtration. Thisprocess is described more fully in Examples 6 and 7 hereinafter.

Coatings were made on the following substrates:

A cellulose fibre board 280 g m² ; (Hermiboard BO16 from CascadesBlendecques SA)

B natural regenerated cellulose film 18 μm ("Cellophane");

C compression moulded starch.

Coating was by means of a large K hand coater (R K Print-CoatInstruments Limited, Royston UK) using a metering rod giving a 12 μm wetcoat thickness. The coated substrates were dried in an air circulatingoven at temperatures and for times set out in the tables below. Somewere treated further by calendering at 130° C. through rollers. Othersamples were dried or further treated by infra-red heating.

Test Methods

MVTR ("Moisture Vapour Transition Rate")

This was measured at 23° C., 85% RH (temperate) or 38° C. 90% RH(tropical) using a Lyssey L80 instrument calibrated daily against astandard PET film. The coated substrate samples were conditioned at roomtemperature for at least 24 h before starting MVTR measurement.Measurements were made at intervals until equilibrium was reached: thefinal 10 readings were averaged.

COBB test

Apparatus conforming to ASTM D2045-64T was used. In this apparatus aweighed coated board sample is clamped coating side upwards to the endof a metal cylinder and de-ionised water is poured into the cylinder. At30 min the water is poured off and the sample is detached, wiped andweighed. The test was carried out at room temperature. The amount ofwater adsorbed is expressed in g per m².

GLOSS

A Microtry Gibbs instrument was used, with a 60 degree incident lightangle.

Grease Resistance

This was determined by the "Kit Test" UM557 as described in 1991 TAPPIUseful Methods Page 175. In this test the test specimen is placed on aflat surface and dripped with each of 12 solutions of castor oil,toluene and heptane differing in composition so as to provide agradation of aggressiveness to hydrophobic materials. Results are shownin the following Tables in which coating thicknesses are approximate.

                  TABLE 1                                                         ______________________________________                                        Effect of coating and drying conditions for board:                                               Cobb values g/m.sup.2 vs                                     Coating drying conditions                                                   Coating   thickness                                                                              Oven       Infra-                                                                              Infra-                                      conditions μm dry 130 C. red red                                           μm wet approx 10 min 10 sec 15 sec                                       ______________________________________                                        1 × 6                                                                             2.4      94         77    60                                          2 × 6  4.8 28 5 3                                                       1 × 12 4.8 53 34 12                                                     2 × 12 9.6 12 1 3                                                     ______________________________________                                    

It is evident that two thin coatings are more effective than a singlecoating of double the thickness. Infra-red heating is both moreeffective and more rapid in giving an impermeable coating. The bestproducts are equal to those obtained using non-biodegradable polymercoating for example:

    ______________________________________                                                       Thickness dry,                                                   approx, μm Cobb g/m.sup.2                                                ______________________________________                                        Acrylic latex    5          5                                                   Viclan (RTM) PVDC 5 1                                                       ______________________________________                                    

The samples whether calendered or infra-red heated showed a high levelof gloss (70-75% at 60 degree light angle) and grease resistance (kitvalue 12/12; not affected by 90 toluene +100 heptane v/v mixture).

                  TABLE 2                                                         ______________________________________                                        MVTR : Comparison with melt coated board                                        (2 × 6 μm applications; 15 sec infra-red drying                               Dry thick- Cobb   Temperate MVTR                                     ness μm g/m.sup.2 g/m.sup.2 /day                                         ______________________________________                                        No Coating 0          137    450                                                2 × 6 μm wet 4.8 3 143                                               melt coated 6.2 5 150                                                       ______________________________________                                    

It is evident that by using the specified latex and drying by infra-red,the level of moisture resistance is substantially equal to that obtainedby melt coating.

                  TABLE 3                                                         ______________________________________                                        Water Vapour Transmission of Coated Cellulose Film                                 Polymer, thickness μm                                                                            MVTR g/m.sup.2 day.sup.-1                          (nominal)              Temperate                                                                              Tropical                                      ______________________________________                                                   0           735      1202                                            PHBV 4.4 148  764                                                             Acrylic 4.8 179 n/a                                                           PVDC 4.8 4 n/a                                                              ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        Coated Starch Substrate: Cobb Test                                                        Cobb Water transmission g/m.sup.2                                 ______________________________________                                        Uncoated    638                                                                 PHBV 9% w/w 516                                                             ______________________________________                                    

EXAMPLE 2 Paper Binder

The same latex was used, but was diluted to 20% w/w for spraying on tothe substrate. The substrate was an 80 g m⁻² air-laid non-bonded paperprecursor. The latex was applied by aerosol spray to both sides of thesubstrate and flowed into the interstices of the paper. The resultingsheets were dried in an oven at 80° C. for 1 h.

Samples from the sheets were tested for peak-load at failure in theseconditions.

Equipment: Instron Type 1122 tensile tester

Sample dimensions: 30 mm×10 mm

Number of replicates: 3, in each of 2 perpendicular directions persample

Test Speed: 5 mm min⁻¹

Jaw Separation: 10 mm

Gauge Length: 10 mm

Full Scale Load: 20N

Results: The mean peak load (N) derived from the 6 tests was reportedfor each sample.

Results are shown in Table 5.

                  TABLE 5                                                         ______________________________________                                                       PHBV % Mean Peak load                                            w/w at failure, N                                                           ______________________________________                                        Unbonded paper precursor                                                                       0        0.9                                                   PHBV-bonded paper 16.5 1.90                                                   precursor                                                                   ______________________________________                                    

This result is to be compared with those reported in the article byMarchessault et al. (TAPPI Journal, May 1993, 76(5), 71-77 at pages73-74):

II Physical and mechanical characteristics of PHB and PHB/HV latexcoated paper

    ______________________________________                                                                          Base                                          Characteristic PHB PHB/BV stock                                             ______________________________________                                        Basis weight g/m.sup.2                                                                      48.0       74.0     37.4                                          Burst index, kPa-m.sup.2 g 0.90 1.65 2.31                                     Breaking length, km 4.75 5.10 6.85                                            Stretch, % 0.95 2.10 1.59                                                     Tensile index, N-m/g 46.30 50.20 67.16                                      ______________________________________                                    

It is evident that the application of the partly crystalline latex usedby these authors does not increase the tear strength of the paper.

EXAMPLE 3

(a) Coatings on paper were made using virgin latex of particle size 0.2μm made by the process of the fourth aspect of the invention (Example 4below), that is, by direct action of chelator and hydrogen peroxide andsurfactant on the fermentation product described in Example 1. Theparticles of the latex contained under 2% w/w of NPCM residues and wereless than 1% w/w crystalline.

The PRA layer was readily coalesced by moderate heating of the latexcoating.

(b) Coatings on board (single application, 12 μm wet thickness) (SeeExample 1) were made using solvent-route* latices of average particlediameter in the range 0.1 to 0.5 μm and dried by various procedures.Table 6 shows the Cobb test results for these latices, with comparisonresults for larger particle virgin latices and for an acrylic latex.

                  TABLE 6                                                         ______________________________________                                        Effect of Particle Size on Cobb Performance                                               Cobb/g m.sup.-2                                                   HV              Oven     IR     Oven   Room                                     Level Particle 130° C. 150° C. 80° C. Temp.                                                    mol % Size μm 3 min 15 min 0.5                                            min overnight                          ______________________________________                                        16     0.1-0.2  9        n.m.   19     9                                        27 0.1 8 14 5 6                                                               27 0.1-0.2 11 n.m. 12 8                                                       23 1.4 108 18 150 n.m.                                                        18 0.9 75 12 157 n.m.                                                         Acrylic  0.08 6  8 6 6                                                      ______________________________________                                         n.m. = not measured                                                           *The solvent route consisted in dissolving the PHA in chloroform to give      5% w/w solution, emulsifying the solution in aqueous sodium                   Nlauroylsarcosinate, removing the chloroform by warming at 65° C.      in a current of nitrogen and adjusting surfactant to ca 5% on PHA. It is      evident that, at particle diameters 0.2μ and under, overnight drying a     room temperature is as effective as at higher temperatures.              

EXAMPLE 4

Preparation of small-particle latex

In a stirred fed batch fermenter Alcaligenes eutrophus was grown onglucose as carbon source, then fermented with glucose and propionic acidunder phosphorus starvation to effect accumulation of a PHAconsisting ofHB and HV units in the molar ratio 85:15.

To the fermenter product at ambient temperature sodiumN-lauroylsarcosinate (6.5% w/w on the PHA) was added and mixed in. Themixture was passed once through a French pressure cell (105.6 kg/cm²) todisrupt the cells, then adjusted to pH 6.8 by addition of 5M potassiumhydroxide. Chelator DTPA (1% w/v), sodium hydrogen phosphates pH 6.8 andsilicone antifoam (100 ppm w/v) were stirred in. The mixture was heatedto 80° C. Hydrogen peroxide 60% w/v solution was added to provide 3% w/vH₂ O₂ in the mixture. The mixture was stirred at 80° C. for 12 h, with afurther hydrogen peroxide addition at 3.5 h to replenish the 3% w/vlevel, and with periodic adjustment of pH to 6.8. The mixture was thenpassed to a tangential flow filter in which soluble material wasseparated, leaving a stable latex of PHA content 400 g/l.

In the resulting latex the average particle size was 0.35 μm. Theparticles were examined by density measurement and found to be less than1% w/w crystalline. The PHA contained less than 2% w/w of NPCM residues.

EXAMPLE 5

Fermenter contents prepared as in Example 4 were treated with lysozyme(0.1% on cell dry weight) at 25° C., pH 7.5 for 5 h. Then the treatmentwith chelator, hydrogen peroxide, and also surfactant (sodiumN-lauroylsarcosinate) and antifoam, and subsequent work-up steps, werecarried out as in Example 4.

The latex average particle size was 0.21 μm and the purity andcrystallinity of the PHA were very similar to those of the latex ofExample 4.

It is evident that the initial treatment at relatively low temperaturehas not led to aggregation of latex particles or to nucleation eventsprovoking crystallisation.

EXAMPLE 6

Conditions for PRA Washing

In a stirred fed batch fermenter Alcaligenes eutrophus was grown onglucose as carbon source, then fermented with glucose and propionic acidunder phosphorus starvation to effect PHBV accumulation. The contents ofthe fermenter were then fed through a wide tube into which high pressuresteam was injected to partly disrupt the cells by heat shock. Theresulting suspension was passed into a first reactor, where it receivedprotease enzyme and was digested for a suitable period. The digestedproduct, consisting mainly of PHA particles and soluble material, wasseparated in an intermittent or continuous centrifuge (A). The particleswere washed with water, back washed to re-suspend them and passed into asecond reactor, where they were stirred with chelator, hydrogen peroxideand surfactant (added in that order) until residual NPCM wassubstantially oxidised. The resulting suspension was passed into anintermittent or continuous centrifuge (B), which was powerful enough todeposit surfactant-coated PHA particles despite the presence ofsurfactant in solution. The deposit in the centrifuge was washed withwater by suspension and further centrifugation and passed into aseparator, which was an intermittent or continuous centrifuge or amembrane flow (MF) filter (C). Here residual soluble material wasremoved, leaving a stable latex.

Heat Shock pH 8.5;

steam temperature 150° C.;

residence time 80 sec;

First Reactor protease concentration 0.5% w/w.

temperature 70° C., residence time 2 h;

Second Reactor pH 7.0,

H₂ O₂ 1.5% w/v, added as 35% w/w aq.soln;

chelator 0.2% w/w DTPA;

surfactant 5.0% w/w,

anti-foam 0.05% w/w;

temperature 80° C., residence time 12 h;

MF-filter varied to give latex polyester concentrations over the range10 to 40% w/w.

The effect of separator conditions on crystallinity, as shown by densitymeasurement, is shown in Table 6.

                  TABLE 6                                                         ______________________________________                                                  Mechanical PHA % w/w in                                               Separator Design product paste Crystallinity                                ______________________________________                                        A         Intermittent                                                           Discharge                                                                     Centrifuge 25 under 1                                                         Continuous                                                                    Discharge                                                                     Centrifuge 25 under 1                                                        B Intermittent                                                                 Discharge                                                                     Centrifuge 25 100                                                             Continuous                                                                    Discharge                                                                     Centrifuge 25 under 1                                                        C Intermittent                                                                 Discharge                                                                     Centrifuge 35 100                                                             Continuous                                                                    Discharge                                                                     Centrifuge 25   under 1 *                                                     MF-filter 40 under 1                                                       ______________________________________                                         * If no solids retention                                                 

Using the continuous discharge centrifuge at B and the membrane flowfilter at C, the following variations were examined.

The surfactants used in successive runs were:

Synperonic A20 (non-ionic: C₁₂ alcohol/20 EO)

Sodium lauryl sarcosinate (anionic). No significant difference wasobserved.

The effect of surfactant concentration was examined in the range 0.5 to8.0% w/w on polymer solids: no difference in crystalline content of thepolyester particles was observed.

The effect of the process on PHBVs of various V content was examined. Atlow V content (5% molar), the particles in the 40% w/w latex were morecrystalline than those in less concentrated latices. At a given latexpolymer concentration, namely at the end of reaction in the secondreactor, a 18% V polyester was under 1% crystalline, compared with a 5%V polyester which was 10-15% crystalline.

In each of the runs the latex average particle size was 0.95 μ for 8% Vor 20% V and 0.55 μ for 16% V. It is believed that these particles areagglomerates of part or all the single particles initially within cellsbut without polyester from other cells. The particles contained lessthan 2% w/w of NPCM residues.

EXAMPLE 7

The initial fermentation was carried out in Example 6.

The contents of the fermenter were fed to a reactor and hydrogenperoxide (4.0% w/v as 35% w/w solution), chelator as in Example 4, andsurfactant (Synperonic A20: C12 linear alcohol 20 EO at 6% w/w) wereadded in that order, with stirring between additions. Silicone anti-foam(0.05% w/w) was also added. The pH was adjusted to 7.0 and thetemperature at 80° C. The mixture was stirred until desorption andsolubilisation of NPCM were complete (12 h), then passed to a membraneflow filter, in which soluble material was separated, leaving a stablelatex of polymer content 40% w/w.

In the resulting latex the average particle size was 0.21 μm. Theparticles were examined by density measurement and found to be less than1% w/w crystalline. They contained less than 2% w/w of NPCM residues.

EXAMPLE 8

PHA latex mixed with acrylic latex

The following starting materials were used:

PHA latex PHBV (23 mol % V) of Mw 1.14×10⁶

solids content 41% w/w

PHA content of particles over 98% w/w

particle size 1.3-1.5 μm (volume average)

crystallinity under 5%

surfactant 3 to 5% w/w on PHA dry solids

origin: fermentation of Alcaligenes eutrophus with glucose andpropionate in growth stage followed by phosphate-limited accumulationstage; harvesting by heat-shock, then digestion with enzyme, hydrogenperoxide and surfactant

Second Latex (% w/w) styrene 25;

2-ethylhexylacrylate 25;

n-butylmethacrylate 43;

methacrylic acid 7;

molecular weight ca 150000

minimum film forming temperature 15-17° C.

solids content 40% w/w

particle size 75-80 nm (0.0075 μm, substantially amorphous

surfactant: sodium lauryl-sulphate

pH 9.0.

Quantities of the two latices were blended to give PHA and acrylic inthe ratio 100 to 15 as dry solids. It was noticed that the blend showedno signs of shear-thickening. The blend was coated on paper boardsamples at 12 μm wet thickness in one application by hand draw-downusing Meyer k-type bars. This wet thickness corresponds to 4-5 μm drythickness. The samples were dried by either of two methods, namely:

oven at 130° C. for 3 min; or

infra-red at 150° C. for 15 sec.

(The 150° C. is the temperature measured at the paper surface). Fivereplicates of samples coated using the blend, and also of samples coatedwith 100% PHA latex or second latex were prepared and subjected to theCobb test as described in Example 1. Results are shown in Table 7.

                  TABLE 7                                                         ______________________________________                                        Latex Composition w/w                                                                          Drying                                                       PHA     ACRYLIC      Method     Cobb g m.sup.-2                               ______________________________________                                                             Oven                                                       100 0 130° C. 3 min  107                                                 IR                                                                          100 0 150° C. 15 sec 27                                                  Oven                                                                        100 15 130° C. 3 min  34                                                 IR                                                                          100 15 150° C. 15 sec 19                                                 Oven                                                                        0 100 130° C. 3 min  6                                                   IR                                                                          0 100 150° C. 15 sec 8                                               ______________________________________                                    

Thus small addition levels of acrylic latex lead to dramatic improvementin film formation at the lower oven drying temperature of 130° C. Thisleads to improved moisture barrier under these conditions.

At the higher drying temperature of 150° C., the effect on Cobbperformance is less marked but still apparent for the acrylic latexblend.

EXAMPLE 9

Stabilisation of PHA latex by acrylic latex

Mixtures of the latices specified in Example 8--in various ratios--weresubjected to shear at 1460 sec⁻¹ at 20° C. in a Bohlin rheometer withcoaxial cylinder geometry. The viscosity of the mixture was measuredover a time period until flocculation took place. Table 8 shows thelengths of this period for 4 mixtures.

                  TABLE 8                                                         ______________________________________                                        % w/w of                                                                        second                                                                        latex 0 1 5 10 15                                                           ______________________________________                                        Floccu-   200 sec  over     over  over  over                                    lation  3500 3500 3500 3500                                                   time  sec sec sec sec                                                       ______________________________________                                    

Using a laboratory stirrer the 5% mixture was found to be stable forover 5 h and the 15% mixture for more than 24 h.

EXAMPLE 10

Latex with steric stabilisation

Example 8 was repeated with the modifications:

(a) a different range of PHA:acrylic ratios was tested; and

(b) each latex contained 3% w/w of "HYPERMER CG6" which is an acrylicgraft copolymer emulsifier formulation in water/propylene glycolcontaining 32% w/w of active agent of HLB number approximately 11-12,available from Imperial Chemical Industries PLC.

Results of the Cobb test are shown in Table 9.

                  TABLE 9                                                         ______________________________________                                        Acrylic Polymer                                                                            Cobb g m.sup.-2                                                  % w/w on solids                                                                            Oven 130° C., 3 min                                                                  IR 150° C., 15s                             ______________________________________                                        0            61            27                                                   4 30 5                                                                        8 13 5                                                                        12 14 4                                                                       16 5 4                                                                        20 5 4                                                                      ______________________________________                                    

With the aid of the emulsifier it is evidently possible to obtain verygood Cobb test behaviour at low levels of the non-biodegradable polymer.

We claim:
 1. A process for making PHA by producing by fermentation abiomass of micro-organism cells containing PHA and harvesting PHA bydecomposing non-PHA cell material (NPCM) by steps including treatmentwith a surfactant and an oxidant, wherein the oxidant is added beforethe surfactant; characterised in that the said treatment is applied tothe biomass before substantial decomposition of the NPCM by other means.2. A process for producing a PHA latex of substantial amorphous contentby:(a) forming a biomass containing micro-organism cells containing PHAparticles, (b) decomposing the non-PHA cell material of the cells tosoluble products; (c) separating the soluble products from the PHAparticles; and (d) forming an aqueous dispersion of PHA particlessubstantially free of such soluble product: characterized by carryingout step (c) by resolving the product of step (b) into a solution of thesoluble products and a non shear-thickening slurry.
 3. A processaccording to claim 2 in which step (c) is carried out by continuouscentrifugation or micro-filtration.
 4. A process according to claim 1,wherein the quantity of the surfactant is in the range of 1% to 7% w/won PHA dry solids.
 5. A process according to claim 1, in which thesurfactant is selected from the group consisting of C₁₂₋₁₆ alkyl/about30 ethoxylate; nonylphenol/about 30 ethoxylate; block copolymer ofethylene oxide and propylene oxide; cetyltrimethylammonium bromide;sodium or ammonium deoxycholate, dodecyl sulphate, N-lauroylsarcosinatedioctyl-sulphosuccinate; and acrylic copolymer emulsifiers.
 6. Theprocess according to claim 1, wherein the harvesting step furthercomprises oxidatively solubilising at least partly the NPCM and applyingto the resulting PHA particles a stabilising quantity of surfactantbefore the PHA has crystallised to the desired extent.